Despite wide-scale use, we have limited understanding of the biotoxicity associated with nanoparticle (NP) exposures which has led to concerns regarding potential adverse health effects. Inhalation is likely a primary exposure route and the association between respired particulate matter with pulmonary disease, underscores the critical need to comprehend how NP impact the lungs. We have a particular interest in single-walled carbon nanotubes (SWNT) as they are widely used and possess a superficial resemblance to asbestos which may be relevant to their long term health consequences. Additional concerns surround the ability of NP to modulate the behavior of infectious agents. Increased susceptibility to infections as a result of NP exposure can have immense consequences particularly for viruses, such as influenza A (IAV), that are notorious for causing global pandemics. As realistic exposure scenarios are likely to involve multiple agents, triggering conserved signaling mechanisms may lead to enhanced detrimental responses that contribute to more severe health outcomes. Based on these notions, the overall objective of this application is to characterize the mechanisms controlling the primary immune response of lung epithelial cells exposed to SWNT and IAV singly and in combination. Our mechanistic focus will center on toll-like receptors (TLRs) as they are an early line of defense to foreign agents that enter in the body. We will test the hypothesis that SWNT of distinct chirality/diameter stimulate TLRs resulting in the production of pro-inflammatory cytokines through activation of the transcription factors NF-k and IRFs. Furthermore, combined exposures of SWNT and IAV will synergistically activate TLR-driven pathways leading to enhanced inflammation and injury. This premise will be tested in 3 comprehensive specific aims which employ state-of-the-art technologies to (1) determine stimulation of distinct TLRs by SWNT having different chiral wrapping angles and diameters as well as by IAV, (2) assess direct interactions of SWNT and IAV and investigate the role of select TLR modulation by these agents in lung epithelial cells and (3) examine the lung inflammatory and clearance response of single and sequential exposures of SWNT and IAV mediated, in part, by TLR-NF-k and/or TLR-IRFs in vivo. In vitro studies will employ lung epithelial cells as a primary target of in vivo exposures, permitting us t define the molecular pathways which lead to organ dysfunction. We will utilize a series of human cell-based TLR screening assays, innovative binding studies and genetically modified mice to address these aims. In addition, we will employ a custom near-infrared fluorescence (NIRF) imaging system to track SWNT in lung tissues and systemically in live animals following SWNT exposures. Results of this work will generate a comprehensive understanding of how multiple aspects of NP affect cell function and will provide reliable in vitro model systems to evaluate and engineer safe nanomaterials. PUBLIC HEALTH RELEVANCE: Nanoparticles, matter having at least one dimension less than 100 nm, are being widely used in industrial, commercial and medical applications. While inhaled ultrafine particles are already associated with pulmonary disease, limited data currently exists regarding the potential effects of nanoparticle exposure on the lung. Additional concerns surround the ability of these particles to modulate the behavior of infectious agents, such as Influenza A virus that is notorious for global pandemics. The work proposed herein will begin to elucidate how normal lung cells and tissues respond to inhaled nanoparticles and Influenza A viruses and results will expose potential long term health consequences that are crucial to the development of improved safety practices and therapeutic regimes.